OP0104 EXPANDED CD8+ T CELL CLONES FROM HLA-B*27-POSITIVE PATIENTS WITH SPONDYLOARTHRITIS SHOW SIGNS OF ANTIGEN-EXPERIENCE
Authors: J. Rademacher, K. Deschler, S. Lacher, A. Huth, M. Utzt, S. Krebs, H. Blum, E. Beltrán, D. Poddubnyy, K. Dornmair
Journal: Annals of the Rheumatic Diseases (2022)

Background The pathogenesis of Spondyloarthritis (SpA) remains unknown but its strong association with some alleles of HLA-B*27 is peculiar. The arthritogenic antigen hypothesis assumes the existence of specific peptides presented by risk-conferring HLA-B*27 alleles to antigen-specific CD8+ T cells, which then initiate or sustain autoimmune reactions. Several studies analyzing T cell receptor (TCR) repertoire found preferred Variable TCR chains and motifs in the hypervariable complementary determining region (CDR) 3, but analyzed only TCR β-chains in bulk analyses 1,2 . Objectives To analyze full sequence information of TCR including matching α- and β-chains from single CD8+ T cells and characterize the transcriptomes of expanded and non-expanded clonotypes in synovial fluid (SF) of SpA patients. Methods We included 17 patients with active gonarthritis: 10 patients with HLA-B27 positive (B27pos) SpA, 4 with HLA-B27 negative (B27neg) SpA and 3 rheumatoid arthritis (RA) patients. Antigen-experienced CD8+ T cells were sorted out of SF by flow cytometry. Single cell sequencing was performed for all patients to analyze matching TCR α- and β-chains. For 7 patients (3 B27pos SpA, 2 B27neg SpA, 2 RA), additionally whole transcriptome analyses were performed. Results We found strong biases when analyzing α and β chains of TCR Variable regions and CDR1 and CDR2 sequences (Figure 1 a,b): AV21, AV12-2, and AV17 were highly enriched in B27pos SpA as compared to B27neg subjects. Amongst the highest expressed clones, we could confirm enrichment for previously described TRBV genes as BV19, BV5-1 and BV6-2. We examined TCR α/β combinations and focused on those detected in at least three different B27pos SpA but not in any of the B27neg patients (Figure 1 c-f). The combinations TRBV19/TRAV21 and TRBV6-2/TRAV21 were most likely specific for B27pos SpA and might reflect interaction of these TCR chains with HLA-B*27. Sequences of CDR3 loops, which predominantly interact with HLA-bound antigenic peptides, revealed striking common structural motifs in α- and β-chains. Focusing on the most prominent TRAV21 chains pairing with TRBV19, 5-1 and 6-2 chains, revealed identical sequences in different patients and striking common structural motifs in α- and β-CDR3 sequences in other patients. Such marked similarities in the antigen-recognition loops of the β-chains associated with TRAV21 suggest common or highly similar antigens. Gene expression levels provided evidence that expanded cell populations had tissue resident memory (TRM) phenotypes (elevated expression of activation, migration and tissue retention markers, downregulated genes characteristic for T cell egress), while this phenotype was not very pronounced in non-expanded cells. Furthermore, markers for T cell exhaustion and apoptosis were elevated in expanded cells of B27pos SpA patients. Figure 1. Distinct TCRαβ V chain usage in expanded clones from HLA-B27 positive SpA patients. A,B Mean number of all productive TRAV (A) and TRBV (B) genes used in expanded, antigen-experienced CD8 T cell clones (&gt;1% of all cells) from SF of 10 B27pos SpA, 4 B27neg SpA and 3 B27neg RA patients. C-F TRAV chains paired with TRBV19 (C), TRBV5-1 (D), TRBV6-2 (E), or TRBV chains paired with TRAV21 (F) with corresponding TRAJ spanning partners in expanded cells (frequency ≥2) from all 10 B27pos SpA. Number of chains are 1250 (C), 886 (D), 1220 (E), and 4006 (F). Conclusion Analysis of single antigen experienced CD8+ T cells from SF of B27pos SpA patients revealed significant clonal expansions and common motifs in the CDR loops. Two of the four CDR1 and CDR2 loops were highly homologous suggesting that these loops interact with α-helices of HLA-B*27. Common motifs in CDR3 loops of expanded clonotypes suggest recognition of a limited set of antigenic peptides presented by HLA-B*27. Many of the expanded clonotypes showed a TRM phenotype, were exhausted and on the way to become apoptotic, which suggests that these clones had sustained contact to specific antigens. References [1]Komech, et al. Rheumatology 2018 [2]Hanson, et al. A&amp;R 2020 Acknowledgements We thank all patients included in this study for their participation. This work was funded by the German Research Foundation (DFG) through grants DO 420/4 to KDo, PO 2124/2-1 to DP, and SyNergy (EXC 2145 SyNergy – ID 390857198) to KDo. Judith Rademacher and Katharina Deschler contributed equally. JR is participant in the BIH-Charité Clinician Scientist Program funded by the Charité –Universitätsmedizin Berlin and the Berlin Institute of Health. The authors would like to thank Martina Seipel for excellent technical assistance, Sabrina Sron for patient recruitment and study coordination, and Hildrun Haibel, Mikhail Protopopov, Fabian Proft, Valeria Rios Rodriguez and Laura Spiller for recruiting patients for this study. Disclosure of Interests None declared